This invention relates to methods and apparatus for assaying biological samples to which a reagent is added and particularly, to computer-controlled methods and apparatus for such assaying.
Assaying processes are well known in which a reagent is added to a sample, and measurements of the sample and reagent are made to identify sample attributes stimulated by the reagent. For example, one such assay process concerns determining in a chromogenic assay the amount of an enzyme present in a biological sample or solution. Such assays are based on the development of a colored product in the reaction solution. The reaction develops as the enzyme catalyzes the conversion of a colorless chromogenic substrate to a colored product.
In such assays it is often required to determine the enzymatic activity of a number of samples and at one or more dilutions. Enzymatic reactions characteristically proceed at a constant rate provided substrate is present in a large molar excess, i.e., the concentration of substrate does not limit the rate of reaction. With such kinetic parameters, it may be convenient to set up several reaction solutions separately in the wells of a microtiter plate, for example, carrying out each reaction for a predetermined constant amount of time and stopping the reactions while they are still in a linear range of the assay. With each of the so-called end-point reactions stopped, no further color development occurs and the reaction solutions in the separate wells of the microtiter may be read at any convenient time.
Plate readers which automatically read the intensity of a colored solution in an array of wells are known. Also, plate readers which measure the amount of fluorescence in a well of a microtiter plate are known.
Classically, assays of the above-described type are performed by a laboratory worker who prepares the sample, manually adds a precise amount of reagent to the sample, and then measures the result at one or more preselected times after the reagent addition. This classical approach is very time consuming for the laboratory worker and additionally when the stimulated reaction yields time-varying results, precise timing on the part of the laboratory worker is required. If such timing is not properly performed, erroneous assay results may occur.
One known laboratory device for partially automating tests to detect fluorescence as a measurable attribute is the Fluoroskan II. The Fluoroskan II includes a plate carrier system to hold a sample-containing plate having a plurality, e.g., 96, of sample-containing wells. A laboratory worker places a portion of the sample into some or all of the wells of the plate, and then adds reagent to the sample-containing wells. The plate is then placed in the Fluoroskan II which automatically measures the fluorescence of the samples in the wells. Although this known apparatus has proven valuable for fluorescence testing, some problems, which were. also inherent in the classical testing, still remain. For example, the addition of reagent by the laboratory worker still requires a large amount of laboratory work time. Also, since the measured results of reactions may be dependent on the time elapsed since reagent addition, the first samples to receive reagent may have progressed past the point of meaningful reaction results by the time all samples have received reagent. Further, some reactions complete so quickly that it is nearly impossible to add reagent to a sample, move the sample plate to the assay apparatus and move the sample to a measurement position before the reaction has run to completion.
A need exists to rapidly screen compounds to determine their effect on a protein""s function such as cell surface proteins like ion channels and receptors, the regulation of which surface proteins can be important in treating certain disease states. Such cell surface proteins permit intracellular transduction of extracellular signals. These cell surface proteins, by transmitting information regarding extracellular stimuli via specific intracellular pathways, induce an appropriate cellular response to external stimuli. The response may be immediate and transient, slow and sustained, or some mixture thereof. By virtue of an array of varied membrane surface proteins, normal (i.e., non-diseased) cells and tissue are exquisitely sensitive to their environment. Compounds which are capable of potentiating or inhibiting activation of voltage-dependent calcium channels are believed to be useful in treating a variety of diseases including certain cardiovascular and nervous system disorders. Similarly, compounds which can affect the functioning of cell surface receptors may be beneficial in the treatment of certain other diseases. Thus, it is desirable to identify compounds capable of activating, potentiating or inhibiting such cell surface proteins using functional assays. Such assays require study of the kinetics of the reaction in real time due to their transient nature. Heretofore, such assays have required a substantial amount of time from highly skilled researchers and technicians to conduct and record the results of individual assays. Also, with respect to functional assays for cell surface proteins which utilize electrophysiological or fluorescence imaging techniques, laboratory equipment which is required is often extremely costly.
A need also exists for an automated analysis apparatus which is capable or automatically adding reagent to contained samples and measuring reaction results to minimize lab worker time requirements, and to provide accurate measurement of time varying reactions. It would also be very advantageous, inasmuch as drug companies now typically screen thousands to tens of thousands of compounds in the hope of finding a desired activity, to be able to provide a rapid automated method for assaying a compound or a series of compounds for ability to affect cell surface proteins, such as ion channels and receptors or cytoplasmic receptors, where a plurality of tests may be conducted seriatim and the results recorded with no, or only occasional, intervention by the researcher
This need is met and a technical advance is achieved with the present invention, which in one aspect is a computer-controlled measurement apparatus for automatically adding reagent to a predetermined sample-containing wells and measuring at least one attribute of the samples in the predetermined wells. The computer-controlled measurement apparatus comprises a plate having a plurality of solution-containing wells, reagent-adding equipment responsive to the computer for adding reagent to the wells, measurement equipment for measuring at least one attribute, e.g., fluorescence, of the solution contained by the wells and moving equipment which is responsive to the computer for aligning the wells with the reagent-adding component and with the fluorescence measurement device.
The computer exercises control over the operation of the various components involved in order to properly coordinate the main functions of the apparatus: reagent addition and attribute measurement. The computer issues several types of commands to provide the necessary coordination. In one embodiment, alignment of wells with reagent-adding and fluorescence-measuring devices is accomplished by computer-issued plate movement commands which are sent to the plate-moving equipment which responds by moving predetermined wells to the reagent-adding position, then to the measurement position. Pump commands are generated by the computer to direct the reagent-adding equipment to add a predetermined volume of the reagent to the predetermined wells at the reagent-adding position. Additionally, measurement commands are generated by the computer to direct the measuring equipment to measure a plurality of fluorescence magnitude values of the samples in the predetermined wells. The values determined by the measurement equipment are recorded within the assay apparatus for later use. In the preferred embodiment, the measured values are first stored within a microcomputer, which comprises the controller, and later moved to a disk drive for long-term storage. The microcomputer may be further equipped with data analysis programs that transform the data into relevant statistics and/or display the data in various formats.
In accordance with one embodiment of the present invention, regarding the performance of the present assay, the controller first aligns a predetermined well containing a sample to be assayed with; the fluid outlet of the reagent-adding equipment, then controls the reagent-adding equipment to add a predetermined volume of reagent to the predetermined well After reagent is added, the predetermined well is aligned, under the control of the controller, with the measurement equipment. The fluorescence of the sample in the predetermined well is measured using a filter and a photomultiplier tube or photodiode array or a charge coupled device (CCD) to detect emitted light, again in response to computer control, while the predetermined well is aligned with the measurement position. Advantageously, the measurement equipment may comprise a light source and filters for stimulating fluorescence and a photomultiplier tube (or photodiode array or CCD) to detect light emitted by the sample in the predetermined well using fiber optic cables to send and receive light.
In certain situations, the sample contained by a well may exhibit background levels of the attribute to be measured, e.g., fluorescence, even before reagent is added to the well., In such cases, it is desirable to measure the attribute prior to adding reagent so that background (pre-reagent) values can be used in data analysis to more accurately evaluate the measured post-reagent values. Accordingly, for certain tests the predetermined well is aligned, under the control of the controller, with the measuring equipment, and pre-reagent measurements are taken and stored.
A method of operation of a computer-controlled fluorescence-measuring apparatus comprises identifying a predetermined well to be measured, moving the predetermined well to a reagent-adding position, and adding reagent to the predetermined well while at the reagent-adding position. After adding reagent, the predetermined well is moved to a measurement position where the fluorescence of the sample in the predetermined well is measured and the data from such measurement recorded. In certain situations, the pre-reagent fluorescence of the sample in the predetermined well may be measured by moving the predetermined well to the measuring position and measuring fluorescence values prior to the adding of reagent.
In a particularly preferred embodiment of the present invention, one or more wells, preferably a plurality of wells arranged in a predetermined array (e.g., a column of 8 wells), are assayed without having to move the plate for addition of reagent once the well or wells to be assayed have been positioned at the reagent-adding/detecting position. Alignment of wells with reagent-adding and fluorescence-measuring devices is accomplished by computer-issued plate movement equipment which responds by moving predetermined wells to such an assay position at which reagent addition and fluorescence measurement both occur. After the assay of the predetermined well(s), other wells may be moved into the assay position, and the method repeated. In such a preferred embodiment, the apparatus may be configured such that fluorescence measurements may desirably be taken from beneath (i.e., through the bottom of) the wells, thus permitting continuous fluorescence measurement before, during and after reagent is pumped into the wells from above.
A further preferred embodiment of the apparatus of the invention employs a computer-controlled robotic arm device to move the position of the fluid outlet(s) of the reagent-adding device between the reagent-adding/detecting position and one or more predetermined positions to pick up aliquots of reagent or buffer for delivery to the wells at the reagent-adding/measurement position or to dispose of spent liquid from, e.g., the wells. This embodiment of the invention provides for the delivery of aliquots of reagent without the need to prime the reagent-adding device with relatively large volumes. Very importantly, employing a computer-controlled robotic arm device to move the reagent-adding device provides the capability of testing several different reagents at the same time, individually delivering a specific reagent to a specific well, and/or sequentially delivering more than one reagent to one or more assay wells. Particularly where the reagent-adding/measurement position accommodates a plurality of wells, a variety of reagents may be added to wells singly, or in duplicate, triplicate, etc.
Further, because the reagent-adding device is capable of picking-up as well as delivering fluid, and because it may be moved under computer control in the x, y, and z axes, in this embodiment of the invention the sample-containing wells may be washed immediately prior to being assayed by automatically aspirating buffer from the assay wells, moving to a discard location and discarding the spent buffer solution, moving to a location having a reservoir of fresh buffer and picking-up an aliquot of the buffer and delivering it to the assay wells (and optionally repeating these steps one or more times) before moving to a location to pick-up an aliquot of reagent and returning to the reagent-adding/detecting position to initiate the assay. In fluoresent indicator-based call assays as described herein, such a washing function may be desirable for removing excess fluorescent indicator which was not incorporated into the cells during loading. Also, by automatically pipetting and discarding buffer, the fluid outlets of the reagent-adding device may be washed between deliveries of different reagents. Similarly, the use of the robotically-controlled reagent-adding device provides for sequential delivery of more than one reagent to any one or more wells being assayed while fluorescence measurements are being taken.
In another embodiment of the present invention, an automated method is provided for testing the response of a cell having receptors or membrane-spanning ion channels to one or more compounds having putative ion channel or receptor modulatory activity, where the ion channels or receptors of the cells, when activated, are capable of directly or indirectly causing a change in the concentration of ions in the cytoplasm, and wherein the degree of activation or inactivation of the ion channels or receptors is determined by a change in fluorescence intensity in the cytoplasm of the cells which cells have been loaded with an amount of an ion-sensitive fluorescent indicator sufficient to detect a change in intracellular ion concentration.
In a particular aspect, the invention provides an automated method for rapid functional screening of compounds to identify potential pharmaceuticals, i.e., drugs. An efficient drug-screening method is provided which utilizes the computer-controlled fluorescence-measuring apparatus described above for rapid automated analysis of one or more compounds that is based on functional evaluation of drug targets, i.e., receptors and ion channels, in their physiological environment, i.e., living cells, in the presence of the potential pharmaceuticals. In the performance of the drug-screening assay, the sample wells contain receptor- and/or ion channel-expressing cells. Where the test compound is a known or putative agonist (a compound that activates the receptor or ion channel) it may be delivered to the wells via the reagent-adding device; where the compound is a known or putative antagonist or a potentiator (that is, an agonist-like compound which augments agonist activity, but cannot itself cause activation, such as the calcium channel potentiator, Bay K8644) the compound may be (1) included in the well(s) before the plate is introduced into the apparatus of the invention, (2) added by the reagent-adding device along with addition of the agonist reagent used to activate the receptors or ion channels, or (3) added by the movable reagent-adding device prior to addition of the agonist (i.e., sequential addition of reagents). Because many cells can be assayed in a relatively short period of time, the present invention enables rapid analysis of replicate samples, including control samples, and provides a possibility for screening multiple compounds and/or multiple doses of compounds in a single operation. Further, because in preferred embodiments, single or sequential additions of compounds can be made without moving the plate, fluorescence changes caused by the addition of a wide variety of known or unknown compounds may be measured, which greatly enhances the ability of the assays to rapidly identify compounds having agonist, antagonist or potentiating activity. In an especially preferred embodiment, the cells are recombinant cells expressing a homogeneous population of recombinant receptors and/or ion channels thereby providing an assay Which is valuable for determining the specificity of a compound having putative agonist or antagonist activity with respect to the receptors or ion channels.